The goal of this project is to study protein folding and stability of subtilisin BPN' and using in vitro mutagenesis, thermodynamic analysis, x- ray crystallography and theoretical methods. The subtilisin system was chosen for its convenience in genetic manipulation, expression, purification and crystallographic characterization of mutant proteins. Mutants which lack proteolytic activity unfold and refold in a highly reversible manner and will be used as vehicles for studying stability mutations by thermodynamic methods. A collection of over 100 mutant subtilisins has been accumulated over the past four years, of which about 20 are significantly more stable that the wild type subtilisin BPN'. Differential scanning calorimetry and denaturation in urea and guanidine- HCI will be used to study the unfolding of mutant subtilisins. The goal is to understand the consequences of specific amino acid changes on the folded, unfolded and transition states in the unfolding process. X-ray crystallography will be used to correlate structural changes in the folded protein with the free energy changes measured by thermodynamic experiments. Various strategies for protein stabilization including attempts to change hydrophobic and electrostatic interactions and decrease chain entropy of the unfolded enzyme will be examined. By studying mutants differing only slightly from the wild type protein, measuring the free energy changes resulting from the modification and correlating specific structural elements to the observed changes in free energy, the theoretical basis for predicting the energetic consequences of a mutation should be improved.